Tricyclic proteasome activity enhancing compounds

ABSTRACT

Proteinopathies result from the proteasome not acting efficiently enough to eliminate harmful proteins and prevent the formation of the pathogenic aggregates. As described herein, inhibition of proteasome-associated deubiquitinase Usp14 results in increased proteasome efficiency. The present invention therefore provides novel compositions and methods for inhibition of Usp14, enhancement of proteasome activity and treatment of proteinopathies.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/811,129, which is the National Stage application ofPCT/US2011/044999, filed Jul. 22, 2011, which claims the benefit ofpriority to U.S. Provisional Patent Application Ser. No. 61/367,173,filed Jul. 23, 2010, the contents of which are hereby incorporated byreference.

GOVERNMENT SUPPORT

This invention was made with U.S. Government support under NationalInstitutes of Health Grant Nos. GM065592, GM66492, and DK082906. Thegovernment has certain rights in the invention.

BACKGROUND

The proteasome is a large protein complex that contains 33 distinctsubunits. Proteasome complexes function as proteases in part to degradeunneeded or misfolded proteins. Proteasomes regulate many aspects ofcell physiology, and proteasome dysfunction has been implicated in avariety of diseases, including cancer and neurodegenerative diseases(Finley D., (2009), Annu Rev. Biochem., 78, 477-513; Hoeller and Dikic,(2009), Nature, 458, 438-444; Demarto and Gillette, (2007), Cell, 129,659-662); Dahlmann, B. (2007) BCB Biochem 8, Suppl 1, S3; Schartz A Land Ciechanover A (2009) Ann Rev Pharmacol Toxicol 49, 73-96).

Most, but not all, proteasome substrates are targeted for degradationvia the covalent attachment of multimeric chains of a small,highly-conserved protein called ubiquitin. Because longer ubiquitinchains interact more strongly with the proteasome than shorter chains(Thrower et al. (2000), EMBO J. 19, 94-102), processes that alterubiquitin chain length frequently also affect substrate degradationrates. The length of ubiquitin chains attached to substrates tagged forproteasome degradation can be modulated by certain proteasome-associateddeubiquitinating enzymes and ubiquitin ligases. These deubiquitinatingenzymes and ligases appear to regulate proteasome activity bydisassembling or extending proteasome-bound ubiquitin chains.

Mammalian proteasomes contain three major deubiquitinating enzymes:Rpn11, Uch37, and Usp14 (Finley D., (2009), Annu Rev. Biochem., 78,477-513). Rpn11 removes ubiquitin from the tagged substrate by cuttingat the junction between the ubiquitin chain and the substrate. Becausethe Rpn11-mediated cleavage occurs following a substrate's commitment toproteolysis, but prior to substrate degradation, Rpn11 helps to preventubiquitin from being degraded along with the substrate, thus minimizingfluctuations in cellular ubiquitin levels. Additionally, because theproteasome substrate must pass through a narrow translocation channelbefore encountering the proteasome's sequestered proteolytic sites,removal of a bulky ubiquitin chain may also facilitate substratetranslocation. Thus, removal of the ubiquitin chain by Rpn11 promotessubstrate degradation through en bloc removal of the ubiquitin chain ata relatively late step in the proteasome pathway (Verma et al., (2002)Science, 298, 611-615; Yao and Cohen, (2002), Nature, 419, 403-407).

In contrast to Rpn11, Uch37 functions prior to the commitment of asubstrate to proteasome degradation. Uch37 disassembles ubiquitin chainsat the substrate-distal tip (Lam et al., (1997), Nature, 385, 737-740),and its enzymatic activity shortens chains rather than remove thementirely. It has been proposed that chain trimming by Uch37 increasesthe ability of the proteasome to discriminate between long and shortmultiubiquitin chains (Lam et al., (1997), Nature, 385, 737-740). Littleis known about how Uch37 may regulate proteasome function in cells.

Very little is known about the function of Usp14. However, the yeastortholog of Usp14, Ubp6, has been suggested to disassemble ubiquitinchains at the substrate-distal tip and to function prior to thecommitment of a substrate to proteasome degradation. (Hanna et al.,(2006), Cell, 127(7), 1401-1413). Ubp6 is thought to act as a proteasomeinhibitor, and prior work on Ubp6 has indicated a noncatalytic mode ofproteasome inhibition (Hanna et al., (2006), Cell, 127(7), 1401-1413).

SUMMARY

The present invention provides compositions and methods for theinhibition of Usp14, the enhancement of proteasome activity and thetreatment of proteinopathies and other diseases for which enhancedprotein breakdown may be therapeutic. Aside from proteinopathies, theenhancement of proteasome activity may be therapeutic for any diseasecharacterized by deficient proteasome activity, or deficient activity ofother components of the ubiquitin-proteasome pathway, such as in vonHippel-Lindau disease, spinocerebellar ataxia 1, Angelman syndrome,giant axon neuropathy, inclusion body myopathy with Paget disease ofbone and frontotemporal dementia (IBMPFD), and others (Lehman, N. L.,(2009), Acta Neuropathologica, 118(3), 329-347; Weihl et al., (2007),Neuromuscular Disorders, 17, 87-87). Enhancing proteasome activity couldalso be therapeutic for diseases in which proteasome substrates areinvolved and contribute to pathology, but which do not satisfy a strictdefinition of proteopathies. For example, numerous oncoproteins areproteasome substrates and their ability to promote cancer couldpotentially be attenuated by enhancing proteasome activity.

One aspect of the invention relates to a compound represented by formulaI,

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,chemically-protected form, enantiomer or stereoisomer thereof; wherein,independently for each occurrence, W, X, Y, Z¹, Z², Z³ and n are asdefined below.

Another aspect of the invention relates to a method of inhibiting thedeubiquitination activity of a Usp14 protein comprising contacting theUsp14 protein with a compound of formula I, or pharmaceuticallyacceptable salt, solvate, hydrate, prodrug, chemically-protected form,enantiomer or stereoisomer thereof.

Another aspect of the invention relates to a method of enhancing proteindegradation by a proteasome in a cell comprising contacting the cellwith a compound of formula I, or pharmaceutically acceptable salt,solvate, hydrate, prodrug, chemically-protected form, enantiomer orstereoisomer thereof.

Another aspect of the invention relates to a method of treating orpreventing a proteinopathy in a subject comprising administering to thesubject a compound of formula I, or pharmaceutically acceptable salt,solvate, hydrate, prodrug, chemically-protected form, enantiomer orstereoisomer thereof, or a pharmaceutical composition comprising thesame.

Another aspect of the invention relates to a method of enhancingproteasome function in a subject comprising administering to the subjecta compound of formula I, or pharmaceutically acceptable salt, solvate,hydrate, prodrug, chemically-protected form, enantiomer or stereoisomerthereof, or a pharmaceutical composition comprising the same.

Another aspect of the invention relates to a method of increasingdegradation of Tau, TDP-43 or ataxin-3 in a subject comprisingadministering to the subject a compound of formula I, orpharmaceutically acceptable salt, solvate, hydrate, prodrug,chemically-protected form, enantiomer or stereoisomer thereof, or apharmaceutical composition comprising the same.

Additional aspects, embodiments, and advantages of the invention arediscussed below in detail. Moreover, the foregoing information and thefollowing detailed description are merely illustrative examples ofvarious aspects and embodiments of the invention, and are intended toprovide an overview or framework for understanding the nature andcharacter of the claimed aspects and embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts two panels: panel A shows an immunoblot that wasperformed using either recombinant Usp14 protein (Purified Usp14) oraffinity-purified Usp14 deficient human proteasomes (Human Proteasome)and anti-Usp14 antibody. The band corresponding to Usp14 is indicated;and panel B shows an immunoblot that was performed using anti-Uch37antibody and Usp14-deficient purified human proteasomes (26S) eitheruntreated (−VS) or treated with Ub-VS (+VS). The band corresponding toUch37 is indicated. Nonspecific bands are indicated with an asterisk.

FIG. 2 depicts three panels: panel A shows a nondenaturing gel analysisthat had undergone in-gel suc-LLVY-AMC staining (indicating presence ofproteasomes) that was performed using commercially available humanproteasomes (Biomol), untreated, purified Usp14 deficient humanproteasomes (−Ub-VS) or Ub-VS treated purified Usp14 deficient humanproteasomes (+Ub-VS); panel B shows a Coomassie Brilliant Blue (CBB)staining of purified, recombinant wild-type Usp14 (Usp14-wt) orcatalytically inactive mutant Usp14 (Usp14-C114A) either with or withouta GST tag, along with a GST control (GST) and a protein size marker(Marker); and panel C shows the results of a gel-shift assay ofproteasomes alone (−), GST and proteasomes (GST), GST tagged wild-typeUsp14 and proteasomes (GST-Usp14-wt), GST tagged catalytically inactivemutant Usp14 and proteasomes (GST-Usp14-C114A), untagged wild-type Usp14and proteasomes (Usp14-wt) or untagged catalytically inactive mutantUsp14 and proteasomes (Usp14-C114A) that had either been stained with ingel suc-LLVY-AMC staining (top, to show the presence of proteasomes) orCoomassie Brilliant Blue (CBB) staining.

FIG. 3 shows the results of a Ub-AMC hydrolysis assay for Usp14 activityin the presence of Ub-VS treated human proteasomes.

FIG. 4 depicts four panels: panel A shows a plot of the linear kinetics(R²>0.99) of the initial rates of Ub-AMC hydrolysis by Usp14 andproteasome at 1 μM Ub-AMC, 1 nM proteasome, and the indicatedconcentration of Usp14; panel B shows a Michaelis-Menten plot ofUsp14-dependent Ub-AMC hydrolysis in the presence of human proteasomefor 25 minutes at 1 μM Ub-AMC, 1 nM proteasome, and the indicatedconcentration of Usp14; panel C shows a plot of the linear kinetics(R²>0.99) of the initial rates of Ub-AMC hydrolysis by Usp14 andproteasome at 4 nM Usp14, 1 nM proteasome and the indicatedconcentration of Ub-AMC; and panel D shows a Michaelis-Menten plot ofconcentration-dependent Ub-AMC hydrolysis in the presence of Usp14 andhuman proteasome for 30 minutes at 4 nM Usp14, 1 nM proteasome and theindicated concentration of Ub-AMC.

FIG. 5 shows an immunoblot that was performed using an antibody specificfor Cyclin B, which also detects polyubiquitinated Cyclin B(Ub_(n)-C1bB). In this experiment, Ub_(n)-C1bB was treated with 26Shuman proteasome alone, human proteasome and wild-type Usp14 (Usp14-wt)or human proteasome and catalytically inactive Usp14 (Usp14-CA), andsubsequently analyzed by immunoblotting.

FIG. 6 depicts two panels: panel A shows a diagram of human Usp14,depicting the ubiquitin-like domain (UBL), the catalytic domain (CAT),the location of exon 4 and the position of Cys114; and panel B showsimmunoblots that were performed on cellular lysates from human 293 cellsthat co-expressed Tau along with either wild-type Usp14 (Usp14-wt),catalytically inactive Usp14 (Usp14-CA), short form Usp14 (Usp14-SF) orUBL domain deficient Usp14 (Usp14-ΔUBL) and stained using antibodiesspecific for Tau, Usp14 or Actin, as indicated.

FIG. 7 shows immunoblots that were performed on cellular lysates from293 cells that co-expressed the indicated forms of flag-tagged Usp14along with tagged hRpn11 either before (Input) or after (IP:26SProteasome) proteasome affinity purification and stained using anti-Flagantibody.

FIG. 8 depicts two panels: panel A shows a statistical plot of thehigh-throughput large scale compound screening for inhibitors of Usp14catalytic inhibitors; and panel B shows a frequency distribution curveused to determine AMC quenching compounds.

FIGS. 9A-9C depict a table of selected compounds of the invention,including percent inhibition values (measured at 8 μM).

FIG. 10 shows the effectiveness of IU2-6 in promoting Tau degradation.Immunoblots were performed using lysates of MEF cells that co-expressedTau and Usp14 and that were treated with 0, 25, 50, 75 or 100 μM IU2-6and stained with antibodies specific for either Tau or Actin.

FIG. 11 depicts an immunoblot showing that IU2-6 acceleratesproteasome-mediated degradation of Ub_(n)-Sic1^(PY) in vitro. Theimmunoblot is probed with anti-T7. The T7 epitope tag is on theSic1^(PY) protein. IU1 is used as a positive control; IU2-6 iscomparable in its effect. Lanes marked “−” has proteasome added but noUsp14.

FIG. 12 depicts five panels: panel A depicts the ¹H NMR spectrum (300MHz, CDCl₃) of IU2-8, panel B depicts the ¹H NMX spectrum (300 MHz,CDCl₃) of IU2-9, panel C depicts the ¹H NMX spectrum (300 MHz, CDCl₃) ofIU2-10, panel D depicts the ¹H NMX spectrum (300 MHz, CDCl₃) of IU2-12and panel E depicts the ¹H NMX spectrum (300 MHz, CDCl₃) of IU2-13.

DETAILED DESCRIPTION

Proteinopathies are a class of diseases and disorders that result fromthe aggregation of abnormal or misfolded proteins. Often, and perhapstypically, such proteins are eliminated from cells throughproteasome-mediated degradation. However, in the case ofproteinopathies, the proteasome does not act efficiently enough toeliminate all of the harmful proteins and prevent the formation of thepathogenic aggregates.

As is demonstrated herein, under normal growth conditions, theproteasome is subject to tonic inhibition brought about by the trimmingof substrate-bound ubiquitin chains by Usp14. Ubiquitin chain trimminginhibits the proteasome because it removes from proteasome substratesthe signal (a ubiquitin chain) that allows recognition by theproteasome; the proteasome-bound substrate can therefore escape withoutbeing degraded. Consequently, an inhibitor of chain trimming by Usp14promotes protein degradation by the proteasome. Thus, as a result ofthis inhibitory mechanism, the mammalian proteasome pathway does notordinarily operate at full efficiency because the pathway is partiallyinhibited by Usp14.

The methods and compositions of the present invention enhance proteasomeactivity by inhibiting the deubiquitinase activity of Usp14. Asdemonstrated herein, this enhanced proteasome activity increases theability of a cell to eliminate abnormal or misfolded proteins, includingthose associated with human disease. The methods and compositions of thepresent invention are therefore useful for the enhancement of proteasomefunction and the treatment of proteinopathies.

Definitions

In order for the present invention to be more readily understood,certain terms and phrases are defined below and throughout thespecification.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e., “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of” “only one of,” or“exactly one of” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

The definition of each expression, e.g., alkyl, m, n, and the like, whenit occurs more than once in any structure, is intended to be independentof its definition elsewhere in the same structure. For example, the R⁵sin the definition of X may be the same or different; and for eachoccurrence of X, each of the R⁵s or R³ (depending on the X) isindependently selected for each occurrence.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., a compound whichdoes not spontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction.

The term “substituted” is also contemplated to include all permissiblesubstituents of organic compounds. In a broad aspect, the permissiblesubstituents include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic substituents oforganic compounds. Illustrative substituents include, for example, thosedescribed herein below. The permissible substituents may be one or moreand the same or different for appropriate organic compounds. Forpurposes of this invention, the heteroatoms such as nitrogen may havehydrogen substituents and/or any permissible substituents of organiccompounds described herein which satisfy the valences of theheteroatoms. This invention is not intended to be limited in any mannerby the permissible substituents of organic compounds.

The term “lower” when appended to any of the groups listed belowindicates that the group contains less than seven carbons (i.e. sixcarbons or less). For example “lower alkyl” refers to an alkyl groupcontaining 1-6 carbons, and “lower alkenyl” refers to an alkenyl groupcontaining 2-6 carbons.

The term “saturated,” as used herein, pertains to compounds and/orgroups which do not have any carbon-carbon double bonds or carbon-carbontriple bonds.

The term “unsaturated,” as used herein, pertains to compounds and/orgroups which have at least one carbon-carbon double bond orcarbon-carbon triple bond.

The term “aliphatic,” as used herein, pertains to compounds and/orgroups which are linear or branched, but not cyclic (also known as“acyclic” or “open-chain” groups).

The term “cyclic,” as used herein, pertains to compounds and/or groupswhich have one ring, or two or more rings (e.g., spiro, fused, bridged).

The term “aromatic” refers to a planar or polycyclic structurecharacterized by a cyclically conjugated molecular moiety containing4n+2 electrons, wherein n is the absolute value of an integer. Aromaticmolecules containing fused, or joined, rings also are referred to asbicylic aromatic rings. For example, bicyclic aromatic rings containingheteroatoms in a hydrocarbon ring structure are referred to as bicyclicheteroaryl rings.

The term “hydrocarbon” as used herein refers to an organic compoundconsisting entirely of hydrogen and carbon.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

The term “heteroatom” as used herein is art-recognized and refers to anatom of any element other than carbon or hydrogen. Illustrativeheteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur andselenium.

The term “alkyl” means an aliphatic or cyclic hydrocarbon radicalcontaining from 1 to 12 carbon atoms. Representative examples of alkylinclude, but are not limited to, methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl,neopentyl, n-hexyl, 2-methylcyclopentyl, and 1-cyclohexylethyl.

The term “substituted alkyl” means an aliphatic or cyclic hydrocarbonradical containing from 1 to 12 carbon atoms, substituted with 1, 2, 3,4, or 5 substituents independently selected from the group consisting ofalkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy,alkenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy,fluoroalkyloxy, sulfhydryl, alkylthio, haloalkylthio, fluoroalkylthio,alkenylthio, alkynylthio, sulfonic acid, alkylsulfonyl,haloalkylsulfonyl, fluoroalkylsulfonyl, alkenylsulfonyl,alkynylsulfonyl, alkoxysulfonyl, halo alkoxysulfonyl,fluoroalkoxysulfonyl, alkenyloxysulfonyl, alkynyloxysulfonyl,aminosulfonyl, sulfinic acid, alkylsulfinyl, haloalkylsulfinyl,fluoroalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, alkoxysulfinyl,haloalkoxysulfinyl, fluoroalkoxysulfinyl, alkenyloxysulfinyl,alkynyloxysulfiny, aminosulfinyl, formyl, alkylcarbonyl,haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl,fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,alkylcarbonyloxy, halo alkylcarbonyloxy, fluoroalkylcarbonyloxy,alkenylcarbonyloxy, alkynylcarbonyloxy, alkylsulfonyloxy, haloalkylsulfonyloxy, fluoroalkylsulfonyloxy, alkenylsulfonyloxy,alkynylsulfonyloxy, halo alkoxysulfonyloxy, fluoroalkoxysulfonyloxy,alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulfinyloxy,haloalkylsulfinyloxy, fluoroalkylsulfinyloxy, alkenylsulfinyloxy,alkynylsulfinyloxy, alkoxysulfinyloxy, halo alkoxysulfinyloxy,fluoroalkoxysulfinyloxy, alkenyloxysulfinyloxy, alkynyloxysulfinyloxy,aminosulfinyloxy, amino, amido, aminosulfonyl, aminosulfinyl, cyano,nitro, azido, phosphinyl, phosphoryl, silyl and silyloxy.

The term “cycloalkyl” is a subset of alkyl which refers to cyclichydrocarbon radical containing from 4 to 15, 4 to 10, or 4 to 7 carbonatoms. Representative examples of cycloalkyl include, but are notlimited to, cyclopentyl and cyclobutyl.

The term “alkylene” is art-recognized, and as used herein pertains to abidentate moiety obtained by removing two hydrogen atoms of an alkylgroup, as defined above.

The term “alkenyl” as used herein means a straight or branched chainhydrocarbon containing from 2 to 10 carbons and containing at least onecarbon-carbon double bond formed by the removal of two hydrogens.Representative examples of alkenyl include, but are not limited to,ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl,5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

The term “alkynyl” as used herein means a straight or branched chainhydrocarbon group containing from 2 to 10 carbon atoms and containing atleast one carbon-carbon triple bond. Representative examples of alkynylinclude, but are not limited, to acetylenyl, 1-propynyl, 2-propynyl,3-butynyl, 2-pentynyl, and 1-butynyl.

The term “carbocyclyl” as used herein means monocyclic or multicyclic(e.g., bicyclic, tricyclic, etc.) hydrocarbons containing from 3 to 12carbon atoms that is completely saturated or has one or more unsaturatedbonds, and for the avoidance of doubt, the degree of unsaturation doesnot result in an aromatic ring system (e.g. phenyl). Examples ofcarbocyclyl groups include 1-cyclopropyl, 1-cyclobutyl, 2-cyclopentyl,1-cyclopentenyl, 3-cyclohexyl, 1-cyclohexenyl and 2-cyclopentenylmethyl.

The term “heterocyclyl”, as used herein include non-aromatic, ringsystems, including, but not limited to, monocyclic, bicyclic (e.g. fusedand spirocyclic) and tricyclic rings, which can be completely saturatedor which can contain one or more units of unsaturation, for theavoidance of doubt, the degree of unsaturation does not result in anaromatic ring system, and have 3 to 12 atoms including at least oneheteroatom, such as nitrogen, oxygen, or sulfur. For purposes ofexemplification, which should not be construed as limiting the scope ofthis invention, the following are examples of heterocyclic rings:azepines, azetidinyl, morpholinyl, oxopiperidinyl, oxopyrrolidinyl,piperazinyl, piperidinyl, pyrrolidinyl, quinuclidinyl, thiomorpholinyl,tetrahydropyranyl and tetrahydrofuranyl. The heterocyclyl groups of theinvention are substituted with 0, 1, 2, 3, 4 or 5 substituentsindependently selected from the group consisting of alkyl, alkenyl,alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkenyloxy,alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy, fluoroalkyloxy,sulfhydryl, alkylthio, haloalkylthio, fluoroalkylthio, alkenylthio,alkynylthio, sulfonic acid, alkylsulfonyl, haloalkylsulfonyl,fluoroalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, alkoxysulfonyl,halo alkoxysulfonyl, fluoroalkoxysulfonyl, alkenyloxysulfonyl,alkynyloxysulfonyl, aminosulfonyl, sulfinic acid, alkylsulfinyl,haloalkylsulfinyl, fluoroalkylsulfinyl, alkenylsulfinyl,alkynylsulfinyl, alkoxysulfinyl, haloalkoxysulfinyl,fluoroalkoxysulfinyl, alkenyloxysulfinyl, alkynyloxysulfiny,aminosulfinyl, formyl, alkylcarbonyl, haloalkylcarbonyl,fluoroalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy,alkoxycarbonyl, haloalkoxycarbonyl, fluoroalkoxycarbonyl,alkenyloxycarbonyl, alkynyloxycarbonyl, alkylcarbonyloxy, haloalkylcarbonyloxy, fluoroalkylcarbonyloxy, alkenylcarbonyloxy,alkynylcarbonyloxy, alkylsulfonyloxy, halo alkylsulfonyloxy,fluoroalkylsulfonyloxy, alkenylsulfonyloxy, alkynylsulfonyloxy, haloalkoxysulfonyloxy, fluoroalkoxysulfonyloxy, alkenyloxysulfonyloxy,alkynyloxysulfonyloxy, alkylsulfinyloxy, haloalkylsulfinyloxy,fluoroalkylsulfinyloxy, alkenylsulfinyloxy, alkynylsulfinyloxy,alkoxysulfinyloxy, halo alkoxysulfinyloxy, fluoroalkoxysulfinyloxy,alkenyloxysulfinyloxy, alkynyloxysulfinyloxy, aminosulfinyloxy, amino,amido, aminosulfonyl, aminosulfinyl, cyano, nitro, azido, phosphinyl,phosphoryl, silyl, silyloxy, and any of said substituents bound to theheterocyclyl group through an alkylene moiety (e.g. methylene).

The term “N-heterocyclyl” as used herein is a subset of heterocyclyl, asdefined herein, which have at least one nitrogen atom through which theN-heterocyclyl moiety is bound to the parent moiety. Representativeexamples include pyrrolidin-1-yl, piperidin-1-yl, piperazin-1-yl,hexahydropyrimidin-1-yl, morpholin-1-yl, 1,3-oxazinan-3-yl and6-azaspiro[2.5]oct-6-yl. As with the heterocyclyl groups, theN-heterocyclyl groups of the invention are substituted with 0, 1, 2, 3,4 or 5 substituents independently selected from the group consisting ofalkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy,alkenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy,fluoroalkyloxy, sulfhydryl, alkylthio, haloalkylthio, fluoroalkylthio,alkenylthio, alkynylthio, sulfonic acid, alkylsulfonyl,haloalkylsulfonyl, fluoroalkylsulfonyl, alkenylsulfonyl,alkynylsulfonyl, alkoxysulfonyl, haloalkoxysulfonyl,fluoroalkoxysulfonyl, alkenyloxysulfonyl, alkynyloxysulfonyl,aminosulfonyl, sulfinic acid, alkylsulfinyl, haloalkylsulfinyl,fluoroalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, alkoxysulfinyl,haloalkoxysulfinyl, fluoroalkoxysulfinyl, alkenyloxysulfinyl,alkynyloxysulfiny, aminosulfinyl, formyl, alkylcarbonyl,haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl,fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,alkylcarbonyloxy, halo alkylcarbonyloxy, fluoroalkylcarbonyloxy,alkenylcarbonyloxy, alkynylcarbonyloxy, alkylsulfonyloxy, haloalkylsulfonyloxy, fluoroalkylsulfonyloxy, alkenylsulfonyloxy,alkynylsulfonyloxy, halo alkoxysulfonyloxy, fluoroalkoxysulfonyloxy,alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulfinyloxy, haloalkylsulfinyloxy, fluoroalkylsulfinyloxy, alkenylsulfinyloxy,alkynylsulfinyloxy, alkoxysulfinyloxy, halo alkoxysulfinyloxy,fluoroalkoxysulfinyloxy, alkenyloxysulfinyloxy, alkynyloxysulfinyloxy,aminosulfinyloxy, amino, amido, aminosulfonyl, aminosulfinyl, cyano,nitro, azido, phosphinyl, phosphoryl, silyl, silyloxy, and any of saidsubstituents bound to the N-heterocyclyl group through an alkylenemoiety (e.g. methylene).

The term “aryl,” as used herein means a phenyl group, naphthyl oranthracenyl group. The aryl groups of the present invention can beoptionally substituted with 1, 2, 3, 4 or 5 substituents independentlyselected from the group consisting of alkyl, alkenyl, alkynyl, halo,haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkenyloxy, alkynyloxy,carbocyclyloxy, heterocyclyloxy, haloalkoxy, fluoroalkyloxy, sulfhydryl,alkylthio, haloalkylthio, fluoroalkylthio, alkenylthio, alkynylthio,sulfonic acid, alkylsulfonyl, haloalkylsulfonyl, fluoroalkylsulfonyl,alkenylsulfonyl, alkynylsulfonyl, alkoxysulfonyl, haloalkoxysulfonyl,fluoroalkoxysulfonyl, alkenyloxysulfonyl, alkynyloxysulfonyl,aminosulfonyl, sulfinic acid, alkylsulfinyl, haloalkylsulfinyl,fluoroalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, alkoxysulfinyl,haloalkoxysulfinyl, fluoroalkoxysulfinyl, alkenyloxysulfinyl,alkynyloxysulfiny, aminosulfinyl, formyl, alkylcarbonyl,haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl,fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,alkylcarbonyloxy, halo alkylcarbonyloxy, fluoroalkylcarbonyloxy,alkenylcarbonyloxy, alkynylcarbonyloxy, alkylsulfonyloxy, haloalkylsulfonyloxy, fluoroalkylsulfonyloxy, alkenylsulfonyloxy,alkynylsulfonyloxy, halo alkoxysulfonyloxy, fluoroalkoxysulfonyloxy,alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulfinyloxy, haloalkylsulfinyloxy, fluoroalkylsulfinyloxy, alkenylsulfinyloxy,alkynylsulfinyloxy, alkoxysulfinyloxy, halo alkoxysulfinyloxy,fluoroalkoxysulfinyloxy, alkenyloxysulfinyloxy, alkynyloxysulfinyloxy,aminosulfinyloxy, amino, amido, aminosulfonyl, aminosulfinyl, cyano,nitro, azido, phosphinyl, phosphoryl, silyl, silyloxy, and any of saidsubstituents bound to the heterocyclyl group through an alkylene moiety(e.g. methylene).

The term “arylene,” is art-recognized, and as used herein pertains to abidentate moiety obtained by removing two hydrogen atoms of an arylring, as defined above.

The term “arylalkyl” or “aralkyl” as used herein means an aryl group, asdefined herein, appended to the parent molecular moiety through an alkylgroup, as defined herein. Representative examples of aralkyl include,but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, and2-naphth-2-ylethyl.

The term “biaryl,” as used herein means an aryl-substituted aryl, anaryl-substituted heteroaryl, a heteroaryl-substituted aryl or aheteroaryl-substituted heteroaryl, wherein aryl and heteroaryl are asdefined herein. Representative examples include 4-(phenyl)phenyl and4-(4-fluorophenyl)pyridinyl.

The term “heteroaryl” as used herein include aromatic ring systems,including, but not limited to, monocyclic, bicyclic and tricyclic rings,and have 3 to 12 atoms including at least one heteroatom, such asnitrogen, oxygen, or sulfur. For purposes of exemplification, whichshould not be construed as limiting the scope of this invention:azaindolyl, benzo(b)thienyl, benzimidazolyl, benzofuranyl, benzoxazolyl,benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzoxadiazolyl,furanyl, imidazolyl, imidazopyridinyl, indolyl, indolinyl, indazolyl,isoindolinyl, isoxazolyl, isothiazolyl, isoquinolinyl, oxadiazolyl,oxazolyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridinyl,pyrimidinyl, pyrrolyl, pyrrolo[2,3-d]pyrimidinyl,pyrazolo[3,4-d]pyrimidinyl, quinolinyl, quinazolinyl, triazolyl,thiazolyl, thiophenyl, tetrahydroindolyl, tetrazolyl, thiadiazolyl,thienyl, thiomorpholinyl, triazolyl or tropanyl. The heteroaryl groupsof the invention are substituted with 0, 1, 2, 3, 4 or 5 substituentsindependently selected from the group consisting of alkyl, alkenyl,alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkenyloxy,alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy, fluoroalkyloxy,sulfhydryl, alkylthio, haloalkylthio, fluoroalkylthio, alkenylthio,alkynylthio, sulfonic acid, alkylsulfonyl, haloalkylsulfonyl,fluoroalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, alkoxysulfonyl,halo alkoxysulfonyl, fluoroalkoxysulfonyl, alkenyloxysulfonyl,alkynyloxysulfonyl, aminosulfonyl, sulfinic acid, alkylsulfinyl,haloalkylsulfinyl, fluoroalkylsulfinyl, alkenylsulfinyl,alkynylsulfinyl, alkoxysulfinyl, haloalkoxysulfinyl,fluoroalkoxysulfinyl, alkenyloxysulfinyl, alkynyloxysulfiny,aminosulfinyl, formyl, alkylcarbonyl, haloalkylcarbonyl,fluoroalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy,alkoxycarbonyl, haloalkoxycarbonyl, fluoroalkoxycarbonyl,alkenyloxycarbonyl, alkynyloxycarbonyl, alkylcarbonyloxy, haloalkylcarbonyloxy, fluoroalkylcarbonyloxy, alkenylcarbonyloxy,alkynylcarbonyloxy, alkylsulfonyloxy, halo alkylsulfonyloxy,fluoroalkylsulfonyloxy, alkenylsulfonyloxy, alkynylsulfonyloxy, haloalkoxysulfonyloxy, fluoroalkoxysulfonyloxy, alkenyloxysulfonyloxy,alkynyloxysulfonyloxy, alkylsulfinyloxy, haloalkylsulfinyloxy,fluoroalkylsulfinyloxy, alkenylsulfinyloxy, alkynylsulfinyloxy,alkoxysulfinyloxy, halo alkoxysulfinyloxy, fluoroalkoxysulfinyloxy,alkenyloxysulfinyloxy, alkynyloxysulfinyloxy, aminosulfinyloxy, amino,amido, aminosulfonyl, aminosulfinyl, cyano, nitro, azido, phosphinyl,phosphoryl, silyl, silyloxy, and any of said substituents bound to theheteroaryl group through an alkylene moiety (e.g. methylene).

The term “heteroarylene,” is art-recognized, and as used herein pertainsto a bidentate moiety obtained by removing two hydrogen atoms of aheteroaryl ring, as defined above.

The term “heteroarylalkyl” or “heteroaralkyl” as used herein means aheteroaryl, as defined herein, appended to the parent molecular moietythrough an alkyl group, as defined herein. Representative examples ofheteroarylalkyl include, but are not limited to, pyridin-3-ylmethyl and2-(thien-2-yl)ethyl.

The term “halo” or “halogen” means —Cl, —Br, —I or —F.

The term “haloalkyl” means an alkyl group, as defined herein, wherein atleast one hydrogen is replaced with a halogen, as defined herein.Representative examples of haloalkyl include, but are not limited to,chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and2-chloro-3-fluoropentyl.

The term “fluoroalkyl” means an alkyl group, as defined herein, whereinall the hydrogens are replaced with fluorines.

The term “hydroxy” as used herein means an —OH group.

The term “alkoxy” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through an oxygen atom.Representative examples of alkoxy include, but are not limited to,methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, andhexyloxy. The terms “alkenyloxy”, “alkynyloxy”, “carbocyclyloxy”, and“heterocyclyloxy” are likewise defined.

The term “haloalkoxy” as used herein means an alkoxy group, as definedherein, wherein at least one hydrogen is replaced with a halogen, asdefined herein. Representative examples of haloalkoxy include, but arenot limited to, chloromethoxy, 2-fluoroethoxy, trifluoromethoxy, andpentafluoroethoxy. The term “fluoroalkyloxy” is likewise defined.

The term “aryloxy” as used herein means an aryl group, as definedherein, appended to the parent molecular moiety through an oxygen. Theterm “heteroaryloxy” as used herein means a heteroaryl group, as definedherein, appended to the parent molecular moiety through an oxygen. Theterms “heteroaryloxy” is likewise defined.

The term “arylalkoxy” or “arylalkyloxy” as used herein means anarylalkyl group, as defined herein, appended to the parent molecularmoiety through an oxygen. The term “heteroarylalkoxy” is likewisedefined. Representative examples of aryloxy and heteroarylalkoxyinclude, but are not limited to, 2-chlorophenylmethoxy,3-trifluoromethyl-phenylethoxy, and 2,3-dimethylpyridinylmethoxy.

The term “sulfhydryl” or “thio” as used herein means a —SH group.

The term “alkylthio” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through a sulfur.Representative examples of alkylthio include, but are not limited,methylthio, ethylthio, tert-butylthio, and hexylthio. The terms“haloalkylthio”, “fluoroalkylthio”, “alkenylthio”, “alkynylthio”,“carbocyclylthio”, and “heterocyclylthio” are likewise defined.

The term “arylthio” as used herein means an aryl group, as definedherein, appended to the parent molecular moiety through an sulfur. Theterm “heteroarylthio” is likewise defined.

The term “arylalkylthio” or “aralkylthio” as used herein means anarylalkyl group, as defined herein, appended to the parent molecularmoiety through an sulfur. The term “heteroarylalkylthio” is likewisedefined.

The term “sulfonyl” as used herein refers to —S(═O)₂— group.

The term “sulfonic acid” as used herein refers to —S(═O)₂OH.

The term “alkylsulfonyl” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through a sulfonylgroup, as defined herein. Representative examples of alkylsulfonylinclude, but are not limited to, methylsulfonyl and ethylsulfonyl. Theterms “haloalkylsulfonyl”, “fluoroalkylsulfonyl”, “alkenylsulfonyl”,“alkynylsulfonyl”, “carbocyclylsulfonyl”, “heterocyclylsulfonyl”,“arylsulfonyl”, “aralkylsulfonyl”, “heteroarylsulfonyl” and“heteroaralkylsulfonyl” are likewise defined.

The term “alkoxysulfonyl” as used herein means an alkoxy group, asdefined herein, appended to the parent molecular moiety through asulfonyl group, as defined herein. Representative examples ofalkoxysulfonyl include, but are not limited to, methoxysulfonyl,ethoxysulfonyl and propoxysulfonyl. The terms “haloalkoxysulfonyl”,“fluoroalkoxysulfonyl”, “alkenyloxysulfonyl”, “alkynyloxysulfonyl”,“carbocyclyloxysulfonyl”, “heterocyclyloxysulfonyl”, “aryloxysulfonyl”,“aralkyloxysulfonyl”, “heteroaryloxysulfonyl” and“heteroaralkyloxysulfonyl” are likewise defined.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The term “aminosulfonyl” as used herein means an amino group, as definedherein, appended to the parent molecular moiety through a sulfonylgroup.

The term “sulfinyl” as used herein refers to —S(═O)— group. Sulfinylgroups are as defined above for sulfonyl groups. The term “sulfinicacid” as used herein refers to —S(═O)OH.

The term “oxy” refers to a —O— group.

The term “carbonyl” as used herein means a —C(═O)— group.

The term “thiocarbonyl” as used herein means a —C(═S)— group.

The term “formyl” as used herein means a —C(═O)H group.

The term “alkylcarbonyl” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through a carbonylgroup, as defined herein. Representative examples of alkylcarbonylinclude, but are not limited to, acetyl, 1-oxopropyl,2,2-dimethyl-1-oxopropyl, 1-oxobutyl, and 1-oxopentyl. The terms“haloalkylcarbonyl”, “fluoroalkylcarbonyl”, “alkenylcarbonyl”,“alkynylcarbonyl”, “carbocyclylcarbonyl”, “heterocyclylcarbonyl”,“arylcarbonyl”, “aralkylcarbonyl”, “heteroarylcarbonyl”, and“heteroaralkylcarbonyl” are likewise defined.

The term “carboxy” as used herein means a —CO₂H group.

The term “alkoxycarbonyl” as used herein means an alkoxy group, asdefined herein, appended to the parent molecular moiety through acarbonyl group, as defined herein. Representative examples ofalkoxycarbonyl include, but are not limited to, methoxycarbonyl,ethoxycarbonyl, and tert-butoxycarbonyl. The terms “haloalkoxycarbonyl”, “fluoroalkoxycarbonyl”, “alkenyloxycarbonyl”,“alkynyloxycarbonyl”, “carbocyclyloxycarbonyl”,“heterocyclyloxycarbonyl”, “aryloxycarbonyl”, “aralkyloxycarbonyl”,“heteroaryloxycarbonyl”, and “heteroaralkyloxycarbonyl” are likewisedefined.

The term “alkylcarbonyloxy” as used herein means an alkylcarbonyl group,as defined herein, appended to the parent molecular moiety through anoxygen atom. Representative examples of alkylcarbonyloxy include, butare not limited to, acetyloxy, ethylcarbonyloxy, andtert-butylcarbonyloxy. The terms “haloalkylcarbonyloxy”,“fluoroalkylcarbonyloxy”, “alkenylcarbonyloxy”, “alkynylcarbonyloxy”,“carbocyclylcarbonyloxy”, “heterocyclylcarbonyloxy”, “arylcarbonyloxy”,“aralkylcarbonyloxy”, “heteroarylcarbonyloxy”, and“heteroaralkylcarbonyloxy” are likewise defined.

The term “alkylsulfonyloxy” as used herein means an alkylsulfonyl group,as defined herein, appended to the parent molecular moiety through anoxygen atom. The terms “haloalkylsulfonyloxy”, “fluoroalkylsulfonyloxy”,“alkenylsulfonyloxy”, “alkynylsulfonyloxy”, “carbocyclylsulfonyloxy”,“heterocyclylsulfonyloxy”, “arylsulfonyloxy”, “aralkylsulfonyloxy”,“heteroarylsulfonyloxy”, “heteroaralkylsulfonyloxy”,“haloalkoxysulfonyloxy”, “fluoroalkoxysulfonyloxy”,“alkenyloxysulfonyloxy”, “alkynyloxysulfonyloxy”,“carbocyclyloxysulfonyloxy”, “heterocyclyloxysulfonyloxy”,“aryloxysulfonyloxy”, “aralkyloxysulfonyloxy”,“heteroaryloxysulfonyloxy” and “heteroaralkyloxysulfonyloxy”

The term “amino” as used herein refers to —NH₂ and substitutedderivatives thereof wherein one or both of the hydrogens areindependently replaced with substituents selected from the groupconsisting of alkyl, haloalkyl, fluoroalkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl,alkylcarbonyl, haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, carbocyclylcarbonyl, heterocyclylcarbonyl,arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyland the sulfonyl and sulfinyl groups defined above; or when bothhydrogens together are replaced with an alkylene group (to form a ringwhich contains the nitrogen). Representative examples include, but arenot limited to methylamino, acetylamino, and dimethylamino.

The term “amido” as used herein means an amino group, as defined herein,appended to the parent molecular moiety through a carbonyl.

The term “cyano” as used herein means a —C≡N group.

The term “nitro” as used herein means a —NO₂ group.

The term “azido” as used herein means a —N₃ group.

The term “phosphinyl” as used herein includes —PH₃ and substitutedderivatives thereof wherein one, two or three of the hydrogens areindependently replaced with substituents selected from the groupconsisting of alkyl, haloalkyl, fluoroalkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl,alkoxy, haloalkoxy, fluoroalkyloxy, alkenyloxy, alkynyloxy,carbocyclyloxy, heterocyclyloxy, aryloxy, aralkyloxy, heteroaryloxy,heteroaralkyloxy, and amino.

The term “phosphoryl” as used herein refers to —P(═O)OH₂ and substitutedderivatives thereof wherein one or both of the hydroxyls areindependently replaced with substituents selected from the groupconsisting of alkyl, haloalkyl, fluoroalkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl,alkoxy, haloalkoxy, fluoroalkyloxy, alkenyloxy, alkynyloxy,carbocyclyloxy, heterocyclyloxy, aryloxy, aralkyloxy, heteroaryloxy,heteroaralkyloxy, and amino.

The term “silyl” as used herein includes H₃Si— and substitutedderivatives thereof wherein one, two or three of the hydrogens areindependently replaced with substituents selected from alkyl, haloalkyl,fluoroalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl,heteroaryl, and heteroaralkyl. Representative examples includetrimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS),tert-butyldimethylsilyl (TBS/TBDMS), triisopropylsilyl (TIPS), and[2-(trimethylsilyl)ethoxy]methyl (SEM).

The term “silyloxy” as used herein means a silyl group, as definedherein, is appended to the parent molecule through an oxygen atom.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations.

As used herein, the term “administering” means providing apharmaceutical agent or composition to a subject, and includes, but isnot limited to, administering by a medical professional andself-administering.

As used herein, the phrases “neurodegenerative disorder” and“neurodegenerative disease” refers to a wide range of diseases and/ordisorders of the central and peripheral nervous system, such asneuropathologies, and includes but is not limited to, Parkinson'sdisease, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS),denervation atrophy, otosclerosis, stroke, dementia, multiple sclerosis,Huntington's disease, encephalopathy associated with acquiredimmunodeficiency disease (AIDS), and other diseases associated withneuronal cell toxicity and cell death.

As used herein, the phrase “pharmaceutically acceptable” refers to thoseagents, compounds, materials, compositions, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

As used herein, the phrase “pharmaceutically-acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting an agent from one organ,or portion of the body, to another organ, or portion of the body. Eachcarrier must be “acceptable” in the sense of being compatible with theother ingredients of the formulation and not injurious to the patient.Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

As used herein, the phrase “pharmaceutically-acceptable salts” refers tothe relatively non-toxic, inorganic and organic salts of compounds.

As used herein, the phrase “proteinopathy” refers to any diseaseassociated with the accumulation and/or aggregation of abnormal ormisfolded proteins. Though proteinopathies are frequentlyneurodegenerative diseases, proteinopathies also include diseases ofother tissues, including the liver, muscle and heart, and include somecancers.

As used herein, the term “subject” means a human or non-human animalselected for treatment or therapy.

As used herein, the phrase “subject suspected of having” means a subjectexhibiting one or more clinical indicators of a disease or condition. Incertain embodiments, the disease or condition is cancer, aneurodegenerative disorder or pancreatitis.

As used herein, the phrase “subject in need thereof” means a subjectidentified as in need of a therapy or treatment of the invention.

As used herein, the phrase “therapeutic effect” refers to a local orsystemic effect in animals, particularly mammals, and more particularlyhumans, caused by an agent. The phrases “therapeutically-effectiveamount” and “effective amount” mean the amount of an agent that producessome desired effect in at least a sub-population of cells. Atherapeutically effective amount includes an amount of an agent thatproduces some desired local or systemic effect at a reasonablebenefit/risk ratio applicable to any treatment. For example, certainagents used in the methods of the present invention may be administeredin a sufficient amount to produce a reasonable benefit/risk ratioapplicable to such treatment.

As used herein, the term “treating” a disease in a subject or “treating”a subject having or suspected of having a disease refers to subjectingthe subject to a pharmaceutical treatment, e.g., the administration ofan agent, such that at least one symptom of the disease is decreased orprevented from worsening.

Inhibitors of Usp14

One aspect of the invention relates to a compound represented by formulaI:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,chemically-protected form, enantiomer or stereoisomer thereof; wherein,independently for each occurrence,

W is

or N-heterocyclyl;

Z¹ is

Z² is

Z³ is

Y is

X is

R¹ is alkyl, substituted alkyl, haloalkyl, fluoroalkyl, cycloalkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl,heteroaralkyl, alkylcarbonyl, haloalkylcarbonyl, fluoroalkylcarbonyl,alkenylcarbonyl, alkynylcarbonyl, carbocyclylcarbonyl,heterocyclylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl,heteroaralkylcarbonyl, sufonyl, sulfinyl or —(CH₂)_(m)R⁶;

R² is alkyl, substituted alkyl, haloalkyl, fluoroalkyl, cycloalkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl,heteroaralkyl, alkylcarbonyl, haloalkylcarbonyl, fluoroalkylcarbonyl,alkenylcarbonyl, alkynylcarbonyl, carbocyclylcarbonyl,heterocyclylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl,heteroaralkylcarbonyl, sulfonyl, sulfinyl or —(CH₂)_(m)R⁶;

R³ is hydrogen, alkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl,hydroxy, alkoxy, alkenyloxy, alkynyloxy, carbocyclyloxy,heterocyclyloxy, haloalkoxy, fluoroalkyloxy, sulfhydryl, alkylthio,haloalkylthio, fluoroalkylthio, alkenylthio, alkynylthio, sulfonic acid,alkylsulfonyl, haloalkylsulfonyl, fluoroalkylsulfonyl, alkenylsulfonyl,alkynylsulfonyl, alkoxysulfonyl, haloalkoxysulfonyl,fluoroalkoxysulfonyl, alkenyloxysulfonyl, alkynyloxysulfonyl,aminosulfonyl, sulfinic acid, alkylsulfinyl, haloalkylsulfinyl,fluoroalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, alkoxysulfinyl,haloalkoxysulfinyl, fluoroalkoxysulfinyl, alkenyloxysulfinyl,alkynyloxysulfiny, aminosulfinyl, formyl, alkylcarbonyl,haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl,fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,alkylcarbonyloxy, haloalkylcarbonyloxy, fluoroalkylcarbonyloxy,alkenylcarbonyloxy, alkynylcarbonyloxy, alkylsulfonyloxy,haloalkylsulfonyloxy, fluoroalkylsulfonyloxy, alkenylsulfonyloxy,alkynylsulfonyloxy, haloalkoxysulfonyloxy, fluoroalkoxysulfonyloxy,alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulfinyloxy,haloalkylsulfinyloxy, fluoroalkylsulfinyloxy, alkenylsulfinyloxy,alkynylsulfinyloxy, alkoxysulfinyloxy, haloalkoxysulfinyloxy,fluoroalkoxysulfinyloxy, alkenyloxysulfinyloxy, alkynyloxysulfinyloxy,aminosulfinyloxy, amino, amido, aminosulfonyl, aminosulfinyl, cyano,nitro, azido, phosphinyl, phosphoryl, silyl, silyloxy or —(CH₂)_(m)R⁷;

R⁴ is cycloalkylalkyl, heterocyclylalkyl, aralkyl or heteroaralkyl;

R^(5a) is hydrogen, halo, lower alkyl or lower haloalkyl;

R^(5b) is hydrogen, halo, lower alkyl or lower haloalkyl;

R⁶ is alkyl, haloalkyl, fluoroalkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, alkylcarbonyl,haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, carbocyclylcarbonyl, heterocyclylcarbonyl,arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl,heteroaralkylcarbonyl, sulfonyl or sulfinyl;

R⁷ is alkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy,alkoxy, alkenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy,haloalkoxy, fluoroalkyloxy, sulfhydryl, alkylthio, haloalkylthio,fluoroalkylthio, alkenylthio, alkynylthio, sulfonic acid, alkylsulfonyl,haloalkylsulfonyl, fluoroalkylsulfonyl, alkenylsulfonyl,alkynylsulfonyl, alkoxysulfonyl, haloalkoxysulfonyl,fluoroalkoxysulfonyl, alkenyloxysulfonyl, alkynyloxysulfonyl,aminosulfonyl, sulfinic acid, alkylsulfinyl, haloalkylsulfinyl,fluoroalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, alkoxysulfinyl,haloalkoxysulfinyl, fluoroalkoxysulfinyl, alkenyloxysulfinyl,alkynyloxysulfiny, aminosulfinyl, formyl, alkylcarbonyl,haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl,fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,alkylcarbonyloxy, haloalkylcarbonyloxy, fluoroalkylcarbonyloxy,alkenylcarbonyloxy, alkynylcarbonyloxy, alkylsulfonyloxy,haloalkylsulfonyloxy, fluoroalkylsulfonyloxy, alkenylsulfonyloxy,alkynylsulfonyloxy, haloalkoxysulfonyloxy, fluoroalkoxysulfonyloxy,alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulfinyloxy,haloalkylsulfinyloxy, fluoroalkylsulfinyloxy, alkenylsulfinyloxy,alkynylsulfinyloxy, alkoxysulfinyloxy, haloalkoxysulfinyloxy,fluoroalkoxysulfinyloxy, alkenyloxysulfinyloxy, alkynyloxysulfinyloxy,aminosulfinyloxy, amino, amido, aminosulfonyl, aminosulfinyl, cyano,nitro, azido, phosphinyl, phosphoryl, silyl or silyloxy;

n is 0, 1 or 2; and

m is 1, 2, 3 or 4.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein W is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R¹ is alkyl, cycloalkyl, heterocyclyl,aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, aralkyl,heteroaralkyl or alkylcarbonyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R¹ is alkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R¹ is lower alkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R¹ is methyl, ethyl, isopropyl orisobutyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R¹ is methyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R² is alkyl, cycloalkyl, heterocyclyl,aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, aralkyl orheteroaralkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R² is alkyl, cycloalkyl, aryl, aralkylor alkylcarbonyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R² is lower alkyl, lower cycloalkyl,optionally substituted phenyl, optionally substituted benzyl or loweralkylcarbonyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R² is methyl, ethyl, cyclopropyl,cyclobutyl, cyclopentyl, phenyl, benzyl, or methylcarbonyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein W is N-heterocyclyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein W is azetidin1-yl, pyrrolidin-1-yl,piperidin-1-yl, morpholin-1-yl or N-methyl piperazin-1-yl (eachoptionally substituted with one to five substituents taken from thelisted N-heterocyclyl substituents).

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein Z¹ is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein Z¹ is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein Z² is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein Z² is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein Z³ is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein Z³ is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein Z¹ is

Z² is

and Z³ is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R³ is hydrogen, halo, lower alkyl,lower haloalkyl, cyano, lower alkyloxy, lower haloalkoxy or amino.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R³ is hydrogen.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein Y is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein Y is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein Y is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R⁴ is cycloalkylalkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R⁴ is heterocyclylalkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R⁴ is aralkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R⁴ is heteroaralkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R⁴ is benzyl substituted with 0, 1, 2,3, 4 or 5 substituents independently selected from the group consistingof alkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy,alkoxy, alkenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy,haloalkoxy, fluoroalkyloxy, sulfhydryl, alkylthio, haloalkylthio,fluoroalkylthio, alkenylthio, alkynylthio, sulfonic acid, alkylsulfonyl,haloalkylsulfonyl, fluoroalkylsulfonyl, alkenylsulfonyl,alkynylsulfonyl, alkoxysulfonyl, haloalkoxysulfonyl,fluoroalkoxysulfonyl, alkenyloxysulfonyl, alkynyloxysulfonyl,aminosulfonyl, sulfinic acid, alkylsulfinyl, haloalkylsulfinyl,fluoroalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, alkoxysulfinyl,haloalkoxysulfinyl, fluoroalkoxysulfinyl, alkenyloxysulfinyl,alkynyloxysulfiny, aminosulfinyl, formyl, alkylcarbonyl,haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl,fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,alkylcarbonyloxy, haloalkylcarbonyloxy, fluoroalkylcarbonyloxy,alkenylcarbonyloxy, alkynylcarbonyloxy, alkylsulfonyloxy,haloalkylsulfonyloxy, fluoroalkylsulfonyloxy, alkenylsulfonyloxy,alkynylsulfonyloxy, haloalkoxysulfonyloxy, fluoroalkoxysulfonyloxy,alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulfinyloxy,haloalkylsulfinyloxy, fluoroalkylsulfinyloxy, alkenylsulfinyloxy,alkynylsulfinyloxy, alkoxysulfinyloxy, haloalkoxysulfinyloxy,fluoroalkoxysulfinyloxy, alkenyloxysulfinyloxy, alkynyloxysulfinyloxy,aminosulfinyloxy, amino, amido, aminosulfonyl, aminosulfinyl, cyano,nitro, azido, phosphinyl, phosphoryl, silyl, silyloxy or —(CH₂)_(m)R⁷.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R⁴ is p-methoxybenzyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein X is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein X is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein R⁵ is hydrogen.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein n is 0.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein n is 1.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein n is 2.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein m is 1.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein m is 2.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein m is 3.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein m is 4.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein the compound is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein the compound is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein Z¹ is

Z² is

Z³ is

and Y is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein Z¹ is

Z² is

Z³ is

Y is

R³ is hydrogen; X is

R^(5a) is hydrogen; and R^(5b) is hydrogen.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein Z¹ is

Z² is

Z³ is

Y is

R³ is hydrogen; X is

R^(5a) is hydrogen; R^(5b) is hydrogen; and n is 1.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein Z¹ is

Z² is

Z³ is

and Y is

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein Z¹ is

Z² is

Z³ is

Y is

R³ is hydrogen; R⁴ is aralkyl; X is

R^(5a) is hydrogen; and R^(5b) is hydrogen.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein Z¹ is

Z² is

Z³ is

Y is

R³ is hydrogen; R⁴ is p-methoxybenzyl; X is

R^(5a) is hydrogen; R^(5b) is hydrogen; and n is 1.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein the compound is selected from thegroup consisting of

Many of the compounds of the invention may be provided as salts withpharmaceutically compatible counterions (i.e., pharmaceuticallyacceptable salts). A “pharmaceutically acceptable salt” means anynon-toxic salt that, upon administration to a recipient, is capable ofproviding, either directly or indirectly, a compound or a prodrug of acompound of this invention. A “pharmaceutically acceptable counterion”is an ionic portion of a salt that is not toxic when released from thesalt upon administration to a recipient. Pharmaceutically compatiblesalts may be formed with many acids, including but not limited tohydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thanare the corresponding free base forms.

Acids commonly employed to form pharmaceutically acceptable saltsinclude inorganic acids such as hydrogen bisulfide, hydrochloric,hydrobromic, hydroiodic, sulfuric and phosphoric acid, as well asorganic acids such as para-toluenesulfonic, salicylic, tartaric,bitartaric, ascorbic, maleic, besylic, fumaric, gluconic, glucuronic,formic, glutamic, methanesulfonic, ethanesulfonic, benzenesulfonic,lactic, oxalic, para-bromophenylsulfonic, carbonic, succinic, citric,benzoic and acetic acid, and related inorganic and organic acids. Suchpharmaceutically acceptable salts thus include sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate,terephathalate, sulfonate, xylenesulfonate, phenylacetate,phenylpropionate, phenylbutyrate, citrate, lactate,.beta.-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate and the like salts. Pharmaceutically acceptable acid additionsalts include those formed with mineral acids such as hydrochloric acidand hydrobromic acid, and those formed with organic acids such as maleicacid.

Suitable bases for forming pharmaceutically acceptable salts with acidicfunctional groups include, but are not limited to, hydroxides of alkalimetals such as sodium, potassium, and lithium; hydroxides of alkalineearth metal such as calcium and magnesium; hydroxides of other metals,such as aluminum and zinc; ammonia, and organic amines, such asunsubstituted or hydroxy-substituted mono-, di-, or trialkylamines;dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine;diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkylamines), such as mono-, bis-, or tris-(2-hydroxyethyl)amine,2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine,N,N-di-lower alkyl-N-(hydroxy lower alkyl)-amines, such asN,N-dimethyl-N-(2-hydroxyethyl)amine, or tri-(2-hydroxyethyl)amine;N-methyl-D-glucamine; and amino acids such as arginine, lysine, and thelike.

Certain compounds of the invention and their salts may exist in morethan one crystal form and the present invention includes each crystalform and mixtures thereof.

Certain compounds of the invention and their salts may also exist in theform of solvates, for example hydrates, and the present inventionincludes each solvate and mixtures thereof.

Certain compounds of the invention may contain one or more chiralcenters, and exist in different optically active forms. When compoundsof the invention contain one chiral center, the compounds exist in twoenantiomeric forms and the present invention includes both enantiomersand mixtures of enantiomers, such as racemic mixtures. The enantiomersmay be resolved by methods known to those skilled in the art, forexample by formation of diastereoisomeric salts which may be separated,for example, by crystallization; formation of diastereoisomericderivatives or complexes which may be separated, for example, bycrystallization, gas-liquid or liquid chromatography; selective reactionof one enantiomer with an enantiomer-specific reagent, for exampleenzymatic esterification; or gas-liquid or liquid chromatography in achiral environment, for example on a chiral support for example silicawith a bound chiral ligand or in the presence of a chiral solvent. Itwill be appreciated that where the desired enantiomer is converted intoanother chemical entity by one of the separation procedures describedabove, a further step may be used to liberate the desired enantiomericform. Alternatively, specific enantiomers may be synthesized byasymmetric synthesis using optically active reagents, substrates,catalysts or solvents, or by converting one enantiomer into the other byasymmetric transformation.

When a compound of the invention contains more than one chiral center,it may exist in diastereoisomeric forms. The diastereoisomeric compoundsmay be separated by methods known to those skilled in the art, forexample chromatography or crystallization and the individual enantiomersmay be separated as described above. The present invention includes eachdiastereoisomer of compounds of the invention and mixtures thereof.

Certain compounds of the invention may exist in different tautomericforms or as different geometric isomers, and the present inventionincludes each tautomer and/or geometric isomer of compounds of theinvention and mixtures thereof.

Certain compounds of the invention may exist in different stableconformational forms which may be separable. Torsional asymmetry due torestricted rotation about an asymmetric single bond, for example becauseof steric hindrance or ring strain, may permit separation of differentconformers. The present invention includes each conformational isomer ofcompounds of the invention and mixtures thereof.

Certain compounds of the invention may exist in zwitterionic form andthe present invention includes each zwitterionic form of compounds ofthe invention and mixtures thereof.

The present invention also includes pro-drugs. As used herein the term“pro-drug” refers to an agent which is converted into the parent drug invivo by some physiological chemical process (e.g., a prodrug on beingbrought to the physiological pH is converted to the desired drug form).Pro-drugs are often useful because, in some situations, they may beeasier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent drug is not. Theprodrug may also have improved solubility in pharmacologicalcompositions over the parent drug. An example, without limitation, of apro-drug would be a compound of the present invention wherein it isadministered as an ester (the “pro-drug”) to facilitate transmittalacross a cell membrane where water solubility is not beneficial, butthen it is metabolically hydrolyzed to the carboxylic acid once insidethe cell where water solubility is beneficial. Pro-drugs have manyuseful properties. For example, a pro-drug may be more water solublethan the ultimate drug, thereby facilitating intravenous administrationof the drug. A pro-drug may also have a higher level of oralbioavailability than the ultimate drug. After administration, theprodrug is enzymatically or chemically cleaved to deliver the ultimatedrug in the blood or tissue.

Exemplary pro-drugs upon cleavage release the corresponding free acid,and such hydrolyzable ester-forming residues of the compounds of thisinvention include but are not limited to carboxylic acid substituents(e.g., —C(O)₂H or a moiety that contains a carboxylic acid) wherein thefree hydrogen is replaced by (C₁-C₄)alkyl, (C₂-C₁₂)alkanoyloxymethyl,(C₄-C₉)1-(alkanoyloxy)ethyl, 1-methyl-1-(alkanoyloxy)-ethyl having from5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbonatoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms,1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms,N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms,1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms,3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl,di-N,N—(C₁-C₂)alkylamino(C₂-C₃)alkyl (such as β-dimethylamino ethyl),carbamoyl-(C₁-C₂)alkyl, N,N-di(C₁-C₂)-alkylcarbamoyl-(C₁-C₂)alkyl andpiperidino-, pyrrolidino- or morpholino(C₂-C₃)alkyl.

Other exemplary pro-drugs release an alcohol or amine of a compound ofthe invention wherein the free hydrogen of a hydroxyl or aminesubstituent is replaced by (C₁-C₆)alkanoyloxymethyl,1-((C₁-C₆)alkanoyloxy)ethyl, 1-methyl-1-((C₁-C₆)alkanoyloxy)ethyl,(C₁-C₆)alkoxycarbonyl-oxymethyl, N—(C₁-C₆)alkoxycarbonylamino-methyl,succinoyl, (C₁-C₆)alkanoyl, α-amino(C₁-C₄)alkanoyl, arylactyl andα-aminoacyl, or α-aminoacyl-α-aminoacyl wherein said α-aminoacylmoieties are independently any of the naturally occurring L-amino acidsfound in proteins, —P(O)(OH)₂, —P(O)(O(C₁-C₆)alkyl)₂ or glycosyl (theradical resulting from detachment of the hydroxyl of the hemiacetal of acarbohydrate).

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991). Protected forms of the inventive compounds are included withinthe scope of this invention.

The term “chemically protected form,” as used herein, pertains to acompound in which one or more reactive functional groups are protectedfrom undesirable chemical reactions, that is, are in the form of aprotected or protecting group (also known as a masked or masking group).It may be convenient or desirable to prepare, purify, and/or handle theactive compound in a chemically protected form.

By protecting a reactive functional group, reactions involving otherunprotected reactive functional groups can be performed, withoutaffecting the protected group; the protecting group may be removed,usually in a subsequent step, without substantially affecting theremainder of the molecule. See, for example, Protective Groups inOrganic Synthesis (T. Green and P. Wuts, Wiley, 1991), and ProtectiveGroups in Organic Synthesis (T. Green and P. Wuts; 3rd Edition; JohnWiley and Sons, 1999).

For example, a hydroxy group may be protected as an ether (—OR) or anester (—OC(═O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl(diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl ort-butyldimethylsilyl ether; or an acetyl ester (—OC(═O)CH₃,—OAc).

For example, an aldehyde or ketone group may be protected as an acetalor ketal, respectively, in which the carbonyl group (C(═O)) is convertedto a diether (C(OR)₂), by reaction with, for example, a primary alcohol.The aldehyde or ketone group is readily regenerated by hydrolysis usinga large excess of water in the presence of acid.

For example, an amine group may be protected, for example, as an amide(—NRC(═O)R) or a urethane (—NRC(═O)OR), for example, as: a methyl amide(—NHC(═O)CH₃); a benzyloxy amide (—NHC(═O)OCH₂C₆H₅NHCbz); as a t-butoxyamide (—NHC(═O)OC(CH₃)₃, —NHBoc); a 2-biphenyl-2-propoxy amide(—NHC(═O)OC(CH₃)₂C₆H₄C₆H₅NHBoc), as a 9-fluorenylmethoxy amide(—NHFmoc), as a 6-nitroveratryloxy amide (—NHNvoc), as a2-trimethylsilylethyloxy amide (—NHTeoc), as a 2,2,2-trichloroethyloxyamide (—NHTroc), as an allyloxy amide (—NHAlloc), as a2-(phenylsulfonyl)ethyloxy amide (—NHPsec); or, in suitable cases (e.g.,cyclic amines), as a nitroxide radical.

For example, a carboxylic acid group may be protected as an ester or anamide, for example, as: a benzyl ester; a t-butyl ester; a methyl ester;or a methyl amide.

For example, a thiol group may be protected as a thioether (—SR), forexample, as: a benzyl thioether; or an acetamidomethyl ether(—SCH₂NHC(═O)CH₃).

Pharmaceutical Compositions

The invention provides pharmaceutical compositions comprising inhibitorsof Usp14. In one aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the compounds described above, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. In another aspect, the agents of theinvention can be administered as such, or administered in mixtures withpharmaceutically acceptable carriers and can also be administered inconjunction with other agents. Conjunctive therapy thus includessequential, simultaneous and separate, or co-administration of one ormore compound of the invention, wherein the therapeutic effects of thefirst administered has not entirely disappeared when the subsequentcompound is administered.

As described in detail below, the pharmaceutical compositions of thepresent invention may be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, e.g., those targeted for buccal,sublingual, and systemic absorption, boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; (3) topical application, for example, asa cream, ointment, or a controlled-release patch or spray applied to theskin; (4) intravaginally or intrarectally, for example, as a pessary,cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8)nasally.

As set out above, in certain embodiments, agents of the invention may becompounds containing a basic functional group, such as amino oralkylamino, and are, thus, capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptableacids. These salts can be prepared in situ in the administration vehicleor the dosage form manufacturing process, or through a separate reactionof a purified compound of the invention in its free base form with asuitable organic or inorganic acid, and isolating the salt thus formedduring subsequent purification. Representative salts include thehydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like (see, for example, Berge et al.(1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric, and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic,and the like.

In other cases, the compounds of the present invention may be compoundscontaining one or more acidic functional groups and, thus, are capableof forming pharmaceutically-acceptable salts withpharmaceutically-acceptable bases. These salts can likewise be preparedin situ in the administration vehicle or the dosage form manufacturingprocess, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically-acceptable metal cation, with ammonia,or with a pharmaceutically-acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like (see, for example,Berge et al., supra).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

The formulations of the compounds of the invention may be presented inunit dosage form and may be prepared by any methods well known in theart of pharmacy. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will varydepending upon the host being treated and the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the agent which produces a therapeutic effect.

In certain embodiments, a formulation of the present invention comprisesan excipient, including, but not limited to, cyclodextrins, liposomes,micelle forming agents, e.g., bile acids, and polymeric carriers, e.g.,polyesters and polyanhydrides; and an agent of the present invention. Incertain embodiments, an aforementioned formulation renders orallybioavailable an agent of the present invention.

Methods of preparing these formulations or compositions may include thestep of bringing into association an compound of the present inventionwith the carrier and, optionally, one or more accessory ingredients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol, glycerolmonostearate, and non-ionic surfactants; (8) absorbents, such as kaolinand bentonite clay; (9) lubricants, such a talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-shelled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. Compositions of the invention may also beformulated for rapid release, e.g., freeze-dried. They may be sterilizedby, for example, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

Exemplary formulations comprising agents of the invention are determinedbased on various properties including, but not limited to, chemicalstability at body temperature, functional efficiency time of release,toxicity and optimal dose.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, administration by injection, infusion orinhalation; topical by lotion or ointment; and rectal by suppositories.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Therapeutic Methods of the Invention

The present invention further provides novel therapeutic methods oftreating proteinopathies and other diseases for which enhanced proteinbreakdown may be therapeutic, including neurodegenerative diseases,comprising administering to a subject, (e.g., a subject in needthereof), an effective amount of a compound of the invention.

A subject in need thereof may include, for example, a subject who hasbeen diagnosed with a proteinopathy or a subject who has been treatedfor a proteinopathy, including subjects that have been refractory to theprevious treatment.

The methods of the present invention may be used to treat anyproteinopathy. Examples of such proteinopathies include, but are notlimited to, Alzheimer's disease, cerebral β-amyloid angiopathy, retinalganglion cell degeneration, prion diseases (e.g. bovine spongiformencephalopathy, kuru, Creutzfeldt-Jakob disease, variantCreutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome,fatal familial insomnia) tauopathies (e.g. frontotemporal dementia,Parkinson's disease, progressive supranuclear palsy, corticobasaldegeneration, frontotemporal lobar degeneration), frontotemporal lobardegeneration, amyotrophic lateral sclerosis, Huntington's disease,familial British dementia, Familial Danish dementia, hereditary cerebralhemorrhage with amyloidosis (Icelandic), CADASIL, Alexander disease,Seipinopathies, familial amyloidotic neuropathy, senile systemicamyloidosis, serpinopathies, AL amyloidosis, AA amyloidosis, type IIdiabetes, aortic medial amyloidosis, ApoAI amyloidosis, ApoIIamyloidosis, ApoAIV amyloidosis, familial amyloidosis of the Finishtype, lysozyme amyloidosis, fibrinogen amyloidosis, dialysisamyloidosis, inclusion body myositis/myopathy, cataracts, medullarythyroid carcinoma, cardiac atrial amyloidosis, pituitary prolactinoma,hereditary lattice corneal dystrophy, cutaneous lichen amyloidosis,corneal lactoferrin amyloidosis, corneal lactoferrin amyloidosis,pulmonary alveolar proteinosis, odontogenic tumor amylois, seminalvesical amyloid, cystric fibrosis, sickle cell disease, critical illnessmyopathy, von Hippel-Lindau disease, spinocerebellar ataxia 1, Angelmansyndrome, giant axon neuropathy, inclusion body myopathy with Pagetdisease of bone and frontotemporal dementia (IBMPFD).

In some embodiments, the subject pharmaceutical compositions of thepresent invention will incorporate the substance or substances to bedelivered in an amount sufficient to deliver to a patient atherapeutically effective amount of an incorporated therapeutic agent orother material as part of a prophylactic or therapeutic treatment. Thedesired concentration of the active agent will depend on absorption,inactivation, and excretion rates of the drug as well as the deliveryrate of the compound. It is to be noted that dosage values may also varywith the severity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions. Typically, dosing will be determinedusing techniques known to one skilled in the art.

The dosage of the subject agent may be determined by reference to theplasma concentrations of the agent. For example, the maximum plasmaconcentration (Cmax) and the area under the plasma concentration-timecurve from time 0 to infinity (AUC (0-4)) may be used. Dosages for thepresent invention include those that produce the above values for Cmaxand AUC (0-4) and other dosages resulting in larger or smaller valuesfor those parameters.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular agent employed, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldprescribe and/or administer doses of the agents of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of an agent of the invention will bethat amount of the agent which is the lowest dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above.

If desired, the effective daily dose of the agent may be administered astwo, three, four, five, six or more sub-doses administered separately atappropriate intervals throughout the day, optionally, in unit dosageforms.

The precise time of administration and amount of any particular agentthat will yield the most effective treatment in a given patient willdepend upon the activity, pharmacokinetics, and bioavailability of aparticular agent, physiological condition of the patient (including age,sex, disease type and stage, general physical condition, responsivenessto a given dosage and type of medication), route of administration, andthe like. The guidelines presented herein may be used to optimize thetreatment, e.g., determining the optimum time and/or amount ofadministration, which will require no more than routine experimentationconsisting of monitoring the subject and adjusting the dosage and/ortiming.

While the subject is being treated, the health of the subject may bemonitored by measuring one or more of the relevant indices atpredetermined times during a 24-hour period. All aspects of thetreatment, including supplements, amounts, times of administration andformulation, may be optimized according to the results of suchmonitoring. The patient may be periodically reevaluated to determine theextent of improvement by measuring the same parameters, the first suchreevaluation typically occurring at the end of four weeks from the onsetof therapy, and subsequent reevaluations occurring every four to eightweeks during therapy and then every three months thereafter. Therapy maycontinue for several months or even years, with a minimum of one monthbeing a typical length of therapy for humans. Adjustments, for example,to the amount(s) of agent administered and to the time of administrationmay be made based on these reevaluations.

Treatment may be initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage may be increased bysmall increments until the optimum therapeutic effect is attained. Inaddition, the combined use of an agent that modulates aautotrophy-associated gene product and a second agent, e.g. anotheragent useful for the treatment of the autophagy-related disease, mayreduce the required dosage for any individual agent because the onsetand duration of effect of the different compounds and/or agents may becomplimentary.

One aspect of the invention relates to a method of inhibiting thedeubiquitination activity of a Usp14 protein comprising contacting theUsp14 protein with any one of the aforementioned compounds, or apharmaceutically acceptable salt, solvate, hydrate, prodrug,chemically-protected form, enantiomer or stereoisomer thereof.

Another aspect of the invention relates to a method of enhancing proteindegradation by a proteasome in a cell comprising contacting the cellwith any one of the aforementioned compounds, or a pharmaceuticallyacceptable salt, solvate, hydrate, prodrug, chemically-protected form,enantiomer or stereoisomer thereof.

Another aspect of the invention relates to a method of treating orpreventing a proteinopathy in a subject comprising administering to thesubject any one of the aforementioned compounds, or a pharmaceuticallyacceptable salt, solvate, hydrate, prodrug, chemically-protected form,enantiomer or stereoisomer thereof.

In certain embodiments, the present invention relates to any of theaforementioned methods, wherein the proteinopathy is selected from thegroup consisting of Alzheimer's disease, cerebral β-amyloid angiopathy,retinal ganglion cell degeneration, bovine spongiform encephalopathy,kuru, Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease,Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,frontotemporal dementia, Parkinson's disease, progressive supranuclearpalsy, corticobasal degeneration, frontotemporal lobar degeneration,frontotemporal lobar degeneration, amyotrophic lateral sclerosis,Huntington's disease, familial British dementia, Familial Danishdementia, hereditary cerebral hemorrhage with amyloidosis (Icelandic),CADASIL, Alexander disease, familial amyloidotic neuropathy, senilesystemic amyloidosis, serpinopathies, AL amyloidosis, AA amyloidosis,type II diabetes, aortic medial amyloidosis, ApoAI amyloidosis, ApoIIamyloidosis, ApoAIV amyloidosis, familial amyloidosis of the Finishtype, lysozyme amyloidosis, fibrinogen amyloidosis, dialysisamyloidosis, inclusion body myositis/myopathy, cataracts, medullarythyroid carcinoma, cardiac atrial amyloidosis, pituitary prolactinoma,hereditary lattice corneal dystrophy, cutaneous lichen amyloidosis,corneal lactoferrin amyloidosis, corneal lactoferrin amyloidosis,pulmonary alveolar proteinosis, odontogenic tumor amylois, seminalvesical amyloid, cystic fibrosis, sickle cell disease and criticalillness myopathy.

In certain embodiments, the present invention relates to any of theaforementioned methods, wherein the proteinopathy is Alzheimer'sdisease, frontotemporal lobar degeneration, amyotrophic lateralsclerosis or Machado-Joseph disease.

Another aspect of the invention relates to a method of treating orpreventing a disease, for which enhanced protein breakdown may betherapeutic, in a subject comprising administering to the subject anyone of the aforementioned compounds, or a pharmaceutically acceptablesalt, solvate, hydrate, prodrug, chemically-protected form, enantiomeror stereoisomer thereof, or a pharmaceutical composition thereof.

In certain embodiments, the present invention relates to any of theaforementioned methods, wherein the disease is selected from the groupconsisting of von Hippel-Lindau disease, spinocerebellar ataxia 1,Angelman syndrome, giant axon neuropathy, inclusion body myopathy withPaget disease of bone and frontotemporal dementia (IBMPFD).

Another aspect of the invention relates to a method of enhancingproteasome function in a subject comprising administering to the subjectany one of the aforementioned compounds, or a pharmaceuticallyacceptable salt, solvate, hydrate, prodrug, chemically-protected form,enantiomer or stereoisomer thereof, or a pharmaceutical compositionthereof.

Another aspect of the invention relates to a method of increasingdegradation of Tau, TDP-43 or ataxin-3 in a subject comprisingadministering to the subject any one of the aforementioned compounds, ora pharmaceutically acceptable salt, solvate, hydrate, prodrug,chemically-protected form, enantiomer or stereoisomer thereof, or apharmaceutical composition thereof.

In certain embodiments, the present invention relates to any of theaforementioned methods, wherein said subject is human.

EXAMPLES

The invention now being generally described, will be more readilyunderstood by reference to the following Examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1—Synthesis of Inhibitors

One approach to the synthesis of selected compounds of the invention isshown and described below. ¹H NMR spectra (300 MHz, CDCl₃) of IU2-8,IU2-9, IU2-10, IU2-12 and IU2-13 are shown in FIG. 12A-12E.

Intermediate 1.

To a suspension of sulfur (11.5 g, 359 mmol) and ethyl 2-cyanoacetate(38.7 g, 342 mmol) in 50 mL of ethanol was added cyclohexan-1-one (33.6g, 342 mmol) drop-wise at room temperature, followed by addition ofdiethyl amine (DEA) (25.0 g, 342 mmol), making sure the temperature wasmaintained below 50° C. The resulting mixture was stirred at 45° C. for1 h, before being cooled to 0° C. and filtered. The collected materialwas washed with cold ethanol and dried under vacuum to give Intermediate1 as yellow solid (57.8 g, yield: 75%).

Intermediate 2.

A suspension of intermediate 1 (7.1 g, 31.5 mmol) in formamide (25 mL)was heated to 145° C. overnight. The resulting mixture was allowed tocool to room temperature and filtered. The collected material was washedwith ethyl acetate to provide Intermediate 2 as a light brown solid (5.2g, yield: 80%).

Intermediate 3.

A solution of intermediate 2 (5.2 g, 25.2 mmol) in phosphorusoxychloride (20 mL) was heated at reflux for 3 h. The solution wasconcentrated and 100 mL of ice water was added followed by dropwiseaddition of 10% ammonia hydroxide to pH 8. The resulting mixture wasextracted with dichloromethane (3×50 mL) and the combined organics weredried (sodium sulfate), filtered and concentrated. The crude materialwas purified by flashing chromatography to give Intermediate 3 (4.4 g,yield: 78%).

Compound 4.

A mixture of intermediate 3 (50 mg, 0.22 mmol), dimethylamine (20 mg,0.44 mmol) and triethyl amine (45 mg, 0.44 mmol) in methanol (10 mL) wasstirred at room temperature overnight. The mixture was concentrated andthe residue was purified by preparative TLC to provide the final product4 (45 mg, yield: 88%).

Additional analogs were made by replacing dimethylamine with othersuitable amines in the last step of the synthesis.

Example 2—Usp14 Mediates Substrate Deubiquitination

To test whether Usp14 is a potent inhibitor of human proteasomes, apurification procedure was developed to generate proteasomes that lackdetectible levels of deubiquitinase Usp14 (modified from Wang et al.,(2007), Biochemistry, 46, 3553-3565). Briefly, human proteasomes wereaffinity-purified on a large scale from a stable HEK293 cell lineharboring HTBH-tagged hRpn11. The cells were Dounce-homogenized in lysisbuffer (50 mM NaH₂PO₄ [pH 7.5], 100 mM NaCl, 10% glycerol, 5 mM MgCl₂,0.5% NP-40, 5 mM ATP, and 1 mM DTT) containing protease inhibitors.Lysates were cleared, then incubated with NeutrAvidin agarose resin(Thermo Scientific) overnight at 4° C. The beads were then washed withexcess lysis buffer followed by the wash buffer (50 mM Tris-HCl [pH7.5], 1 mM MgCl₂ and 1 mM ATP). For VS-proteasomes, 1 to 1.5 μM of Ub-VS(Boston Biochem) was added to the resin and incubated at 30° C. for 2 h.Residual Ub-VS was removed by washing the beads with at least 20 bed volof wash buffer. 26S proteasomes were eluted from the beads by cleavage,using TEV protease (Invitrogen).

Using this proteasome purification procedure, human proteasomes wereaffinity-purified from a hRpn11-tagged line of HEK293 cells.Purification of proteasomes lacking Usp14 but containing relateddeubiquitinase Uch37 was confirmed by western blot using an anti-Usp14and anti-Uch37 antibodies (FIGS. 1A and 1B, respectively). The purifiedUsp14-free proteasome (also described as 26S proteasomes) retained highlevels of deubiquitinating activity that could be irreversibly inhibitedby treating the proteasome with ubiquitin-vinylsulfone (Ub-VS, Yao etal., (2006) Nat. Cell Biol., 8, 994-1002). Ub-VS inhibitsdeubiquitination of substrates by forming adducts with the Cys aminoacid located in the active site of thiol protease class deubiquitinatingenzymes. As demonstrated in FIG. 1B, addition of Ub-VS to 26Sproteasomes resulted in enzymatically inactive VS-Uch37 adducts formingwith all detectable Uch37.

In order to generate pure, recombinant Usp14 enzyme, GST-Usp14 (WT andC114A variants) was expressed in E. coli strain Rosetta 2 (DE3) cells(Novagen). Cultures were grown at 37° C. until OD₆₀₀ reached 0.6 to 0.8,and expression was induced overnight with 1 mM IPTG at room temperature.Cells were then harvested in PBS containing protease inhibitors andlysed by French press. The cleared lysates were incubated with GSTSepharose 4B resin (GE Healthcare) at 4° C. for 1 h, and subsequentlywashed with excess PBS, followed by PBS containing 100 mM NaCl. The GSTmoiety was removed by thrombin in the cleavage buffer (50 mM Tris-HCl[pH 8.0], 150 mM NaCl, 2.5 mM CaCl₂, and 0.1% 2-mercaptoethanol) for 3 hat room temperature. GST-tagged Usp14 proteins for proteasome bindingassays were eluted before thrombin cleavage using elution buffer (10 mMreduced glutathione in 50 mM Tris-HCl [pH 8.0]).

The inhibited “VS-proteasomes” described above, which lack endogenousdeubiquitination activity due to Ub-VS treatment, were successfullyreconstituted with recombinant Usp14 (FIG. 2). An Ub-AMC hydrolysisassay was performed with 1 nM of Ub-VS treated human proteasome(VS-Proteasome) alone, 400 nM of Usp14 alone, or VS-proteasome that hadbeen reconstituted with 4 or 40 nM of recombinant Usp14 protein. As hasbeen described above, the deubiquitination activity of the VS-proteasomewas almost completely inhibited (FIG. 3). In contrast, the reconstitutedUsp14/VS proteasome demonstrated substantial deubiquitination activity(FIG. 3). In fact, the Usp14/VS proteasome demonstrated an 800-foldincrease in Ub-AMC hydrolyzing activity over that of isolated Usp14alone (FIG. 3). Thus, the enzymatic activity of Usp14 is increased byits complexing with the proteasome. Therefore, the Ub-AMC assay allowsthe success of reconstitution to be followed.

The Ub-AMC assay was also used to examine the kinetics of Ub-AMChydrolysis by the reconstituted Usp14-proteasome complexes. Ub-AMChydrolysis by Usp14/VS proteasomes that had been reconstituted withvarious amounts of Usp14 was monitored over a period of 30 minutes (FIG.4). Analysis of the results of this assay demonstrated the affinity ofUsp14 for the proteasome is approximately 4 nM.

Example 3—Usp14 Inhibits Proteasomal Degradation

The effect of Usp14 on the degradation of ubiquitinated substrates wasexamined using an in vitro degradation assay using theubiquitin-dependant proteasome substrate polyubiquitinated cyclin B(Ub_(n)-C1nB). In these experiments, Ub_(n)-C1nB was incubated withhuman proteasomes (4 nM), containing either wild-type or catalyticallyinactive Usp14 (60 nM). The catalytically inactive Usp used in theseassays was Usp14-C114A, which contains a mutation in Usp14's active sitefor deubiquitination. Notably, both wild-type Usp and Usp14-C114A areable to bind to 26S mammalian proteasomes (FIG. 2). As demonstrated inFIG. 5, Usp14 strongly inhibits the degradation of cyclin B, while theactive site mutant of Usp14 showed little inhibitory effect. The lack ofinhibition of Ub_(n)-C1nB degradation by the active site mutantindicates that the ubiquitin chain trimming activity of wild-type Usp14is required for Usp14's inhibition of proteasome degradation. Indeed,extensive trimming of the ubiquitin groups from cyclin B was evident byimmunoblot analysis in the samples containing wild-type Usp14, but wasnearly eliminated when catalytically inactive Usp14 was used (FIG. 5).

An effect of Usp14 on Tau degradation in human cells was observed in thehuman cell line, HEK293. Tau was coexpressed with exogenous wild-type orcatalytically inactive Usp14 and Tau protein levels were determined bywestern blot. Expression of wild-type Usp14, but not enzymaticallyinactive Usp14, stabilized Tau in the human cell line (FIG. 6). In fact,expression of enzymatically inactive Usp14 in HEK293 cells resulted inaccelerated Tau degradation (FIG. 6B). This dominant negative effectlikely reflects the displacement of endogenous, wild-type Usp14 from theproteasome. This hypothesis was confirmed using a mutant form of Usp14that lacks the N-terminal UBL domain (Usp14-ΔUBL). The N-terminal UBLdomain (FIG. 6A) is the principal proteasome-binding site on Usp14.Deletion of the UBL attenuated the dominant negative effect (FIG. 7),indicating that proteasome binding is required for the mediation of thiseffect.

The short form (SF) of Usp14 is an endogenous Usp14 splice variant thatis expressed from mRNA that lacks a junctional exon (exon 4) between theN-terminal ubiquitin-like domain of Usp14 and its catalytic domain(Wilson et al., (2002), Nat. Genet., 32, 420-425; FIG. 6A). Like thecatalytically inactive mutant of Usp14, Usp14-SF exhibited a dominantnegative effect on Tau stability in HEK293 cells (FIG. 6A). Thissuggests that Usp14-SF may be an endogenous inhibitor of Usp14.Consistent with this possibility, Usp14-SF is able to bind proteasome,but unlike the wild-type enzyme, it is not activated enzymatically byproteasome binding (FIG. 7).

Example 4—Specific Inhibitors of Usp14

As demonstrated above, chain trimming at the proteasome by Usp14 is akey regulatory step in the ubiquitin-dependent proteolytic pathway.Therefore, in order to identify enhancers of proteasome function, ahigh-throughput screen for small molecule Usp14 inhibitors was performedusing VS-proteasomes reconstituted with recombinant Usp14 and assayedwith Ub-AMC (FIG. 8).

Compounds were screened for Usp14/26S inhibition in 384-well low-volumeplates in duplicate. Data processing was done by a robust Z-score methodand each compound was plotted using Spotfire software. Compounds overthe cut-off of Z>5 were mostly autofluorescent and were therefore notcounted. To exclude quenching compounds that only affect AMCfluorescence, 312 primary hits were tested for quenching of AMC amine,and pure quenchers were scored as false-positives and excluded fromfurther analysis (FIG. 8B). Of the 63,052 compounds analyzed in thehigh-throughput screen, 215 were identified as true inhibitors of Usp14.

In order to identify compounds that specifically inhibited Usp14 butwere not general deubiquitinase inhibitors, the 215 hit compounds werecounterscreened against a panel of deubiquitinating enzymes. Among thehit compounds that inhibited the activity of Usp14 but not any othertested deubiquitinase was IU2-1 (FIGS. 9A-9C).

Example 5—Enhancement of Proteasomal Degradation by IU2-6

IU2-6 was identified as a specific inhibitor of Usp14 (FIGS. 9A-9C). Theability of IU2-6 to enhance proteasome function in living cells wasexamined. Tau was used in this experiment because a number ofneurodegenerative diseases, including Alzheimer's disease, result fromthe pathological aggregation of Tau protein. Tau was expressed in MEFcells, which were then treated with IU2-6 at concentrations from 25 to100 μM. After 36 hours of Tau expression, MEF cells were incubated with0, 25, 50, 75 or 100 μM of IU2-6 for 6 hours. As seen in FIG. 10, IU2-6reduced Tau levels at all concentrations tested.

It was next tested whether IU2-6 could serve as an enhancer ofubiquitinated substrate degradation by the proteasome. The effect ofIU2-6 on the degradation of ubiquitinated substrates was examined usingan in vitro degradation assay using the ubiquitin-dependant proteasomesubstrate polyubiquitinated Sic1^(PY) (Ub_(n)-Sic1^(PY)).Ub_(n)-Sic1^(PY) was incubated with human proteasomes, containing eitherwith or without wild-type Usp14 in the absence or presence of either IU1or IU2-6. As expected, addition of Usp14 to the proteasome complexenhanced chain trimming and dramatically inhibited substratedegradation. Addition of either IU1 or IU2-6 stimulated the activity ofthe Usp14-containing proteasomes in degrading Ub_(n)-Sic1^(PY) andinhibited ubiquitin chain trimming (FIG. 11).

EQUIVALENTS

The present invention provides, in part, methods for the enhancement ofprotein turnover by the proteasome and the treatment of diseasesinvolving either proteasome substrates, upstream components of theubiquitin-proteasome pathway, or the proteasome itself. While specificembodiments of the subject invention have been discussed, the abovespecification is illustrative and not restrictive. Many variations ofthe invention will become apparent to those skilled in the art uponreview of this specification. The appended claims are not intended toclaim all such embodiments and variations, and the full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

We claim:
 1. A method of treating a tauopathy in a subject in needthereof comprising administering to the subject an effective amount of acompound represented by formula I:

wherein, independently for each occurrence, W is

Z¹ is

Z² is

Z³ is

Y is

X is

R¹ is alkyl, substituted alkyl, heterocyclyl, aryl, aralkyl, heteroaryl,heteroaralkyl, heterocyclylalkyl, cycloalkylalkyl, or alkylcarbonyl; R²is alkyl, substituted alkyl, heterocyclyl, aryl, aralkyl, heteroaryl,cycloalkylalkyl, heterocyclylalkyl, heteroaralkyl, or alkylcarbonyl; R³is hydrogen, alkyl, halo, haloalkyl, alkoxy, haloalkoxy, amino, orcyano; R^(5a) is independently hydrogen, halo, lower alkyl or lowerhaloalkyl; R^(5b) is independently hydrogen, halo, lower alkyl or lowerhaloalkyl; and n is 0, 1 or 2; or a pharmaceutically acceptable salt,enantiomer or stereoisomer thereof.
 2. The method of claim 1, whereinthe tauopathy is selected from the group consisting of frontotemporaldementia, Parkinson's disease, progressive supranuclear palsy,corticobasal degeneration, frontotemporal lobar degeneration andAlzheimer's disease.
 3. The method of claim 1, wherein R¹ is alkyl,heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl,aralkyl, heteroaralkyl or alkylcarbonyl.
 4. The method of claim 3,wherein R¹ is a linear or branched alkyl.
 5. The method of claim 1,wherein R² is methyl, ethyl, cyclopropyl, cyclobutyl, cyclopentyl,phenyl, benzyl, or methylcarbonyl.
 6. The method of claim 1, wherein R³is hydrogen, halo, lower alkyl, lower haloalkyl, cyano, lower alkyloxy,lower haloalkoxy or amino.
 7. The method of claim 1, wherein n is
 1. 8.The method of claim 1, wherein the compound is selected from the groupconsisting of


9. The method of claim 1, wherein R¹ is a linear or branched alkyl. 10.The method of claim 9, wherein R² is cycloalkyl or substitutedcycloalkyl.
 11. The method claim 10, wherein R² is cyclopropyl orsubstituted cyclopropyl.
 12. The method of claim 7, wherein R² iscycloalkyl or substituted cycloalkyl.
 13. The method of claim 12,wherein R¹ is alkyl.
 14. The method of claim 13, wherein R¹ is a linearor branched alkyl.
 15. The method of claim 14, wherein R¹ is methyl orethyl.
 16. The method of claim 2, wherein the tauopathy is Parkinson'sdisease.